Attention has re-focused on the potential for genotoxicity and related adverse events in the course of gene therapy as a consequence of leukemias that developed in patients treated with retroviral vector. The potential for genotoxicity from retroviral vectors can be understood on the basis of known integration rates, though subsequent interactions between cell and transgene, as well as secondary mutations, make prediction of outcome complex. In contrast, recombinant adeno-associated virus (rAAV) gene therapy vector integration is not well understood in terms of frequency, mechanism, or possible effects on adjacent chromosmal sequnces. Without an understanding of rAAV integration, it is not possible to estimate the risks of either insertiohal activation or large-scale chromosomal rearrangements. We use a reporter system for rAAV DNA recombination events, which we have previously used to characterize the formation of rAAV episomes, to probe the molecular interactions that lead to rAAV integration. Based on the selfcomplementary rAAV (scAAV) vector, these reporters provide genetic detection, rather than physical detection of DNA products, to give a sensitive and accurate quantification of the fate of the vector genomes. We will continue studies that we have already undertaken in understanding the role of the hairpin terminal repeat (TR) sequences, and their interactions with cellular factors in circularization, concatemerization, and integration in cultured cells (Aim 1). We will apply these vectors and methods to the study of integration in mouse liver tissue, emphasizing the preferential use of specific TR structures and the effects of inhibitors of DNA metabolism on TR-mediated recombinations (Aim 2). We will employ a new assay to determine the rate of rAAV integration in vivo without induction of cell division. Finally, we will use an scAAV vector with insertional activation properties similar to unmodified retrovirus vectors to assay for tumorigenicity in mouse hepatocytes (Aim 3). We expect that this project will provide a new level of detail to our knowledge of rAAV integration and help pave the way to the use of these vectors with a clear understanding of any potential risks. Public: The risk of genotoxicity from rAAV gene therapy vectors is difficult to estimate because the mechanism of integration and the frequency in vivo are unknown. We will use specialized reporter vectors to report what types of DNA recombination events have taken place in the target cells.